(600h) Modulating DNA Nanoarchitectures As a Novel, Biomimetic Method of Controlled Therapeutic Release

Harnessing biology in the design of therapeutic nanocarriers presents a facile way to incorporate profound functionality into novel platforms. Synthetic nucleic acids are ideal candidates for constructing modular nanocarriers due to their inherent structural properties that can be exploited for controllable assembly at the nanoscale. However, no research thus far has focused on exploiting these properties for the controlled release of drugs. Our lab has been a pioneer in controlling drug release from novel DNA nanoparticles by manipulating the biological mechanisms that drugs use to bind to nucleic acids. In this work, controlled release of the intercalating drug daunomycin is achieved by modulating DNA nanoarchitectures to exploit the sequence-specific and cooperative intercalation of the drug.

We compared two DNA sequences, one comprised of a high-affinity intercalation motif (AGC) and one random with low GC-content (RAN). We also investigated the effect of DNA monomer length, corresponding to either 4X, 8X, or 12X intercalating sites (i.e., 12 bp, 24 bp, or 36 bp drug binding region), and surface curvature using planar gold wafers and 15 nm AuNPs. Drug dissolution studies were performed in PBS at 37^oC. Cumulative drug release was plotted over time and converted into a fractional release, (M_t/M_∞), where M_t is mass released at time t and M_∞ is the mass released over an infinite amount of time.

Drug loading depends only on DNA sequence length and is the identical between both sequences at maximum loading. However, release rate is dependent on sequence and monomer length. On planar gold, drug release from RAN sequences with 4X, 8X, and 12X intercalation sites resulted in drug release for up to 96 h, 144 h, and 144 h, respectively, with a linear decrease in release rate. The release from AGC sequences is extended across every length tested compared to the RAN sequence. Increasing the length of AGC monomers to 4X, 8X, and 12X intercalation sites resulted in drug release for up to 144 h, 216 h, and 288 h, respectively. For the AGC monomer with 12X intercalation sites the release profile shows nearly zero-order kinetics. On DNA-AuNPs, the surface curvature induces more rapid drug release. Here, the sequence specific affinity still plays a significant effect. Daunomycin release from AuNP-RAN lasts for 48 h, while AuNP-AGC extends the release to 96 h. Additionally, the release rate constant of AuNP-AGC is half that of AuNP-RAN (0.267 and 0.547, respectively).

The controlled release of daunomycin is driven by the natural binding mechanism of the drug to double-stranded DNA. The pattern of hydrogen bonding that dictates the affinity of intercalation is dependent upon the specific DNA sequence, and we show in this work that incorporating high affinity binding sequences, e.g. the AGC-motif, leads to significantly reduced drug release rates. Additionally, daunomycin intercalation is cooperative and stabilizes DNA duplexes, thus making monomer length play a substantial role in drug release rates. For both sequences, increasing length is inversely correlated with release rate. However, for the AGC sequence, drastic changes in release kinetics are observed as the monomer length increases. On AuNPs, the highly curved surface results in a reduction in DNA density as the drug diffuses away from the particle surface and thus reduces the chance of re-intercalation and the steric restriction of a dense DNA monolayer. The result is a more rapid drug release.

These results show, for the first time, that the natural binding mechanism of daunomycin to DNA can be harnessed to produce controllable drug release. This was achieved by designing a high-affinity binding sequence that exploits the chemical interactions between the drug and the DNA base pairs and extending its length to exploit the cooperative binding and stabilizing of DNA by the drug. Nucleic acid nanocarriers represent the next generation of therapeutics and thus controlling drug release will be imperative. By incorporating specific, drug binding layers within rationally designed particles a highly tailorable platform can be synthesized. Therefore, the results herein will substantially improve the future design and clinical efficacy of novel therapeutic nanocarriers.